Regulatory Elements Associated with CBF Transcription Factors of Maize

ABSTRACT

Compositions and methods for regulating expression of heterologous polynucleotide sequences in a plant are provided. Compositions are novel nucleotide sequences comprising an isolated stress-induced promoter natively linked to the maize CBF1 or CBF2 coding region. A method for expressing a heterologous polynucleotide sequence in a plant using the regulatory sequences disclosed herein is provided. The method comprises transforming a plant cell to comprise a heterologous polynucleotide sequence operably linked to one or more of the regulatory sequences of the present invention and regenerating a stably transformed plant from the transformed plant cell.

CROSS REFERENCE

This utility application claims the benefit of U.S. ProvisionalApplication No. 60/971,278, filed Sep. 11, 2007, which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more particularly to regulation of gene expression in plants.

BACKGROUND OF THE INVENTION

Expression of heterologous DNA sequences in a plant host is dependentupon the presence of operably linked regulatory elements that arefunctional within the plant host. Choice of the regulatory element willdetermine when and where within the organism the heterologous DNAsequence is expressed. Where continuous expression is desired throughoutthe cells of a plant, and/or throughout development, constitutivepromoters are utilized. In contrast, where gene expression in responseto a stimulus is desired, inducible promoters are the regulatory elementof choice. Where expression in specific tissues or organs is desired,tissue-specific or tissue-preferred promoters may be used to driveexpression preferentially in certain tissues or organs. Suchtissue-specific or tissue-preferred promoters may be temporallyconstitutive or inducible. In either case, additional regulatorysequences upstream and/or downstream from a core promoter sequence maybe included in expression constructs of transformation vectors to bringabout varying levels of expression of heterologous nucleotide sequencesin a transgenic plant.

As this field develops and more genes become accessible, a greater needexists for transformed plants with multiple genes, and these multipleexogenous genes typically need to be controlled by separate regulatorysequences. Further, some genes should be regulated constitutively,whereas other genes should be expressed at certain developmental stagesor locations in the transgenic organism. Accordingly, a variety ofregulatory sequences having diverse effects is needed.

Diverse regulatory sequences are also needed as undesirable biochemicalinteractions can result from using the same regulatory sequence tocontrol more than one gene. For example, transformation with multiplecopies of a regulatory element may cause problems, such that expressionof one or more genes may be adversely affected.

Transgenic modulation of early sensing and signaling genes involved inabiotic stress responses requires expression of the transgenes earlyupon exposure to the stress and at a moderate level. Also, expression ofsuch transgenes needs to be turned off at later stages of stressexposure so as to avoid the continued induction of downstream targets, ascenario which can easily lead to yield penalty. The current inventionprovides two regulatory sequences which can be used for early expressionand tight modulation of signaling and sensing genes, for transgenicmodulation of plant stress tolerance.

The inventors herein disclose the isolation and characterization ofpromoters associated with stress-related transcription factors that canserve as regulatory elements for expression of isolated nucleotidesequences of interest, thereby impacting various traits in plants.Alternatively or additionally, the promoters may be used to drivescorable markers.

SUMMARY OF THE INVENTION

The invention provides plant promoters which regulate transcription andare induced in response to abiotic stress.

In an embodiment, the promoter drives transcription in astress-responsive manner, wherein said promoter comprises a nucleotidesequence selected from the group consisting of:

a) sequences natively associated with, and that regulate expression of,DNA coding for the CBF1 or CBF2 transcription factor in maize (Zeamays);

b) the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2;and

c) a sequence comprising a fragment of the nucleotide sequence set forthin SEQ ID NO: 1 or SEQ ID NO: 2.

Further embodiments are to expression cassettes, transformation vectors,plants, plant cells and plant parts comprising the above nucleotidesequences. The invention is further to methods of using the sequence inplants and plant cells. An embodiment of the invention further comprisesthe nucleotide sequences described above operably linked to a detectablemarker.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the regulatory sequence of ZmCBF1 (1329 base pairs).Putative cis-acting elements, including a TATA box (bold), CRT/DREconsensus sequence (boxed with double lines), ABRE core motif (singleunderline), and myc-binding sequences (boxed with single lines) aremarked. Bold letters, double underline and an arrow indicate thetranslation start site, ATG. All features are also indicated in thesequence listing at SEQ ID NO: 1.

FIG. 2 shows the regulatory sequence of ZmCBF2 (2209 base pairs).Putative cis-acting elements, including a TATA box (bold), CRT/DREconsensus sequence (boxed with double lines), and myc-binding sequences(boxed with single line) are marked. A putative transcription start siteis indicated by a double underline. Bold letters, double underline andan arrow indicate the translation start site, ATG. All features are alsoindicated in the sequence listing at SEQ ID NO: 2.

DETAILED DESCRIPTION OF THE INVENTION

All public disclosures referred to herein are incorporated by reference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting.

Plants adapt to environmental stresses such as cold, drought, andsalinity through modulation of gene expression. Promoter regions ofstress-inducible genes may comprise cis-acting elements, which are DNAfragments recognized by trans-acting factors. Transacting factorsinclude plant hormones such as abscisic acid (ABA) which has been shownto bind to an ABA-responsive element (ABRE); see, for example,Yamaguchi-Shinozaki, et al., (2005) Trends in Plant Science 10(2):88-94.Other transacting factors include nuclear proteins capable of binding toregulatory DNA and interacting with other molecules, notably DNAPolymerase III, to initiate transcription of DNA operably linked to saidregulatory DNA. These transcription factors may exist as families ofrelated proteins that share a DNA-binding domain. The transcriptionfactor genes may themselves be induced by stress. Furthermore, thedownstream targets of cis-regulated genes may be transcription factors,creating a complex network of gene response cascades.

DRE/CRT (Dehydration Response Element/C-Repeat) cis elements function inresponse to stress and have been identified in numerous plant species,including Arabidopsis, barley, Brassica, citrus, cotton, eucalyptus,grape, maize, melon, pepper, rice, soy, tobacco, tomato and wheat. TheDRE/CRT elements comprise a core binding site, A/GCCGAC, recognized bythe trans-activating factors known as DREB1 (DRE-Binding) and CBF(C-Repeat Binding Factor). Secondary structure in proximity to the ciselement, and/or multiple cis factors, appear to be additional componentsnecessary for stress-inducible expression. (For reviews see, Agarwal, etal., (2006) Plant Cell Rep 25:1263-1274; Yamaguchi-Shinozaki andShinozaki (2005) Trends in Plant Science 10(2):88-94.) The promoterregions of the CBF/DREB genes may comprise cis-acting elements such asICEr1 and ICEr2 (Zarka, et al., (2003) Plant Physiol. 133:910-918;Massari and Murre (2000) Mol. Cell. Bio. 20:429-440).

Other transcription factors include the MYC and MYC-like proteins (see,for example, Zhu, et al., (2003) J. Biol. Chem. 278(48):47803-47811).

In accordance with the invention, nucleotide sequences are provided thatallow regulation of transcription in response to stress. The sequencesof the invention comprise regulatory elements associated withstress-responsive polynucleotides. Thus, the compositions of the presentinvention comprise novel nucleotide sequences for plant regulatoryelements natively associated with the nucleotide sequences coding forZmCBF1 and ZmCBF2.

ZmCBF1 and ZmCBF2 belong to the DREB1 class of transcription factorswhich are induced early upon exposure to abiotic stresses such as cold,drought, and salt. The promoter of the Arabidopsis CBF3 gene is known tocontain five myc-binding sites and to bind to a basic helix-loop-helixprotein known as the ICE1 (inducer of CBF expression) which is anupstream regulatory protein of CBF. A DREB1B promoter from rice (GenBankEF556551) has been isolated by Gutha and Reddy, and was shown to beinduced by abiotic stress. (Plant Molecular Biology online10.1007/s11103-008-9391-8, 28 Aug. 2008). Badawi, et al., (Plant CellPhysiol. 49(8):1237-1249 (2008)) reported an analysis of wheat ICE(inducer of DREB1/CBF expression) genes and their impact on expressionof cold-regulated genes of Arabidopsis.

Identification of the regulatory regions of ZmCBF1 and ZmCBF2 will (a)allow their use for low-level expression and tight modulation of earlysensing and signaling genes, and (b) help to identify the maizeorthologs of ICE1 transcription factor either by yeast one-hybrid screenor to confirm the function of maize orthologs identified by sequencehomology using electrophoretic mobility shift assays. ZmCBF1 and ZmCBF2were previously published; see, U.S. Pat. Nos. 7,253,000 and 7,317,141.As documented in Example 4 of U.S. Pat. No. 7,317,141, endogenous ZmCBF2is rapidly induced by cold stress. Similar work with ZmCBF1 has shownthat it too is inducible by cold, with peak expression at 4 hours afterimposition of cold stress. ZmCBF1 has also been evaluated for expressionunder drought stress; it is induced within 24 hours of the withholdingof water, with peak expression at 27 hours. In case of gene expressionunder cold, induced levels decreased after the peak and returned to zeroupon recovery from cold over a period of 48 hours. In case of geneexpression under drought, induced levels gradually declined after thepeak expression and returned to zero after about 36 hours of withholdingwater. In both tests, no ZmCBF1 expression was observed prior to thestress treatment. Stress-induced endogenous expression is consistentwith the identification of stress-responsive elements within the ZmCBFpromoter sequences as described elsewhere herein.

The ZmCBF regulatory element will be operably linked to a sequence ofinterest, regulating initiation of transcription of the operably-linkedpolynucleotide, which will provide for modification of the phenotype ofthe plant. Such initiation of transcription may be referred to as“promoter activity.” Such modification includes modulating theproduction of an endogenous product, as to amount, relativedistribution, or the like, or production of an exogenous expressionproduct to provide for a novel function or product. For example, such apromoter is useful for modulation of expression of sequences encodingstress-responsive proteins, including other transcription factors.Additionally, linking a stress-induced promoter with a marker, and, inparticular, a visual marker, may be useful in tracking the expression ofa linked gene of interest.

A method for expressing an isolated nucleotide sequence in a plant usinga regulatory sequence disclosed herein is provided. The method comprisestransforming a plant cell with a transformation vector that comprises anisolated nucleotide sequence operably linked to one or more of the plantregulatory sequences of the present invention and regenerating a stablytransformed plant from the transformed plant cell. In this manner, theregulatory sequences are useful for controlling the expression ofendogenous as well as exogenous products in a stress-induced manner.

Frequently it is desirable to have preferential expression of a DNAsequence in a tissue of an organism, or under certain environmentalconditions. For example, increased resistance of a plant to insectattack might be accomplished by genetic manipulation of the plant'sgenome to comprise a tissue-specific promoter operably linked to aheterologous insecticide gene such that the insect-deterring substancesare specifically expressed in the susceptible plant tissues. Increasedtolerance to abiotic stress might be accomplished by geneticmanipulation of the plant's genome to comprise a stress-induced promoteroperably linked to a heterologous gene encoding a plant hormone suchthat the hormone is specifically expressed under the stress conditions.Preferential expression of the heterologous nucleotide sequence in theappropriate tissue or under the appropriate conditions reduces the drainon the plant's resources that occurs when a constitutive promoterinitiates transcription of a heterologous nucleotide sequence throughoutthe cells of the plant.

Alternatively, it might be desirable to inhibit expression of a nativeDNA sequence within a plant's tissues to achieve a desired phenotype.For example, a hairpin configuration comprising all or a portion of therespective ZmCBF promoter may be used to downregulate the nativestress-responsive CBF1 or CBF2, or to downregulate any other codingregion to which the ZmCBF promoter is linked. When such downregulationof a stress-responsive polynucleotide is appropriately targeted, forexample with a reproductive-tissue-preferred promoter, certain planttissues may avoid detrimental effects of stress. In another example, theZmCBF promoter is operably linked to an antisense nucleotide sequence,such that stress-induced expression of the antisense sequence producesan RNA transcript that interferes with translation of the mRNA of asecond DNA sequence in a subset of the plant's cells.

DEFINITIONS

For the purposes of the present invention, unless indicated otherwise orapparent from the context, a “subject plant” or “subject plant cell” isone in which genetic alteration, such as transformation, has beeneffected as to a gene of interest, or is a plant or plant cell which isdescended from a plant or plant cell so altered and which comprises thealteration. A “control” or “control plant” or “control plant cell”provides a reference point for measuring changes in the subject plant orplant cell.

A control plant or control plant cell may comprise, for example: (a) awild-type plant or plant cell, i.e., of the same genotype as thestarting material for the genetic alteration which resulted in thesubject plant or subject plant cell; (b) a plant or plant cell of thesame genotype as the starting material but which has been transformedwith a null construct (i.e., with a construct which has no known effecton the trait of interest, such as a construct comprising a marker gene);(c) a plant or plant cell which is a non-transformed segregant amongprogeny of a subject plant or subject plant cell; (d) a plant or plantcell genetically identical to the subject plant or subject plant cellbut which is not exposed to conditions or stimuli that would induceexpression of the gene of interest; or (e) the subject plant or subjectplant cell itself, under conditions in which the gene of interest is notexpressed.

By “stress-induced” is intended favored expression under conditions ofstress to the plant, particularly abiotic stress, for example conditionsof drought, cold, high temperature, or high salinity.

By “regulatory element” is intended sequences responsible for expressionof the associated coding sequence including, but not limited to,promoters, terminators, enhancers, introns, and the like.

By “terminator” is intended a regulatory region of DNA that causes RNApolymerase to disassociate from DNA, causing termination oftranscription.

By “promoter” is intended a regulatory region of DNA capable ofregulating the transcription of a sequence linked thereto, i.e., aregion of DNA having promoter activity. It usually comprises a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particular codingsequence.

A promoter may additionally comprise other recognition sequencesgenerally positioned upstream or 5′ to the TATA box, referred to asupstream promoter elements, which influence the transcription initiationrate and further include elements which impact spatial and temporalexpression of the linked nucleotide sequence. It is recognized thathaving identified the nucleotide sequences for the promoter regiondisclosed herein, it is within the state of the art to isolate andidentify further regulatory elements in the 5′ region upstream from theparticular promoter region identified herein. Thus the promoter regiondisclosed herein may comprise upstream regulatory elements such as thoseresponsible for tissue and temporal expression of the coding sequence,and may include enhancers, the DNA response element for atranscriptional regulatory protein, ribosomal binding sites,transcriptional start and stop sequences, translational start and stopsequences, activator sequence and the like.

In the same manner, the promoter elements which enable expression understress conditions can be identified, isolated, and used with other corepromoters. By core promoter is meant the minimal sequence required toinitiate transcription, such as the sequence called the TATA box whichis common to promoters in genes encoding proteins. Thus the upstreampromoter of ZmCBF1 or ZmCBF2 can optionally be used in conjunction withits own or core promoters from other sources. The promoter may be nativeor non-native to the cell in which it is found.

The isolated promoter sequence of the present invention can be modifiedto provide for a range of expression levels of the isolated nucleotidesequence. Less than the entire promoter region can be utilized and theability to drive stress-induced expression retained. It is recognizedthat expression levels of mRNA can be modulated with specific deletionsof portions of the promoter sequence. Thus, the promoter can be modifiedto be a weak or strong promoter. Generally, by “weak promoter” isintended a promoter that drives expression of a coding sequence at a lowlevel. By “low level” is intended levels of about 1/10,000 transcriptsto about 1/100,000 transcripts to about 1/500,000 transcripts.Conversely, a strong promoter drives expression of a coding sequence ata high level, or at about 1/10 transcripts to about 1/100 transcripts toabout 1/1,000 transcripts. Generally, at least about 20 nucleotides ofan isolated promoter sequence will be used to drive expression of anucleotide sequence.

It is recognized that to increase transcription levels enhancers can beutilized in combination with the promoter regions of the invention.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are known in the art and include theSV40 enhancer region, the 35S enhancer element, and the like.

The promoter of the present invention can be isolated from the 5′ regionof its native coding region or 5′ untranslated region (5′ UTR). Likewisethe terminator can be isolated from the 3′ region flanking itsrespective stop codon. The term “isolated” refers to material, such as anucleic acid or protein, which is: (1) substantially or essentially freefrom components which normally accompany or interact with the materialas found in its naturally occurring environment; or (2) if the materialis in its natural environment, the material has been altered bydeliberate human intervention to a composition and/or placed at a locusin a cell other than the locus native to the material. Methods forisolation of promoter regions are well known in the art.

The complete genomic sequence for maize ZmCBF1 gene has been previouslypublished (US Patent Application Publication Number 2006/0162027). Themaize CBF1 promoter is set forth in SEQ ID NO: 1 and is 1373 nucleotidesin length. The maize CBF2 promoter is set forth in SEQ ID NO: 2 and is2266 nucleotides in length.

Motifs identified in the ZmCBF1 or ZmCBF2 promoter are shown in FIG. 1and in the sequence listing.

The promoter regions of the invention may be isolated from any plant,including, but not limited to corn (Zea mays), canola (Brassica napus,Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye(Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower(Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max),tobacco (Nicotiana tabacum), millet (Panicum spp.), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), oats (Avena sativa), barley (Hordeum vulgare), vegetables,ornamentals, and conifers. Preferably, plants include corn, soybean,sunflower, safflower, canola, wheat, barley, rye, alfalfa and sorghum.

Promoter sequences from other plants may be isolated according towell-known techniques based on their sequence homology to the homologouscoding region of the coding sequences set forth herein. In thesetechniques, all or part of the known coding sequence is used as a probewhich selectively hybridizes to other sequences present in a populationof cloned genomic DNA fragments (i.e., genomic libraries) from a chosenorganism. Methods are readily available in the art for the hybridizationof nucleic acid sequences. An extensive guide to the hybridization ofnucleic acids is found in Tijssen, Laboratory Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays”, Elsevier, New York (1993); and CurrentProtocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995).

“Functional variants” of the regulatory sequences are also encompassedby the compositions of the present invention. Functional variantsinclude, for example, the native regulatory sequences of the inventionhaving one or more nucleotide substitutions, deletions or insertions andwhich drive expression of an operably-linked sequence under conditionssimilar to those under which the native promoter is active. Functionalvariants of the invention may be created by site-directed mutagenesis orby induced mutation, or may occur as allelic variants (polymorphisms).

As used herein, a “functional fragment” is a truncated regulatorysequence formed by one or more deletions from a larger regulatoryelement. For example, the 5′ portion of a promoter up to the TATA boxnear the transcription start site can be deleted without abolishingpromoter activity, as described by Opsahl-Sorteberg, H-G., et al.,(2004) “Identification of a 49-bp fragment of the HvLTP2 promoterdirecting aleruone cell specific expression” Gene 341:49-58. Suchfragments should retain promoter activity, particularly the ability todrive stress-induced expression. Activity can be measured by Northernblot analysis, reporter activity measurements when using transcriptionalfusions, and the like. See, for example, Sambrook, et al., (1989)Molecular Cloning: A Laboratory Manual (2nd ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.), herein incorporated by reference.

Functional fragments can be obtained by use of restriction enzymes tocleave the naturally occurring regulatory element nucleotide sequencesdisclosed herein; by synthesizing a nucleotide sequence from thenaturally occurring DNA sequence; or can be obtained through the use ofPCR technology. See particularly, Mullis, et al., (1987) MethodsEnzymol. 155:335-350, and Erlich, ed. (1989) PCR Technology (StocktonPress, New York).

For example, a routine way to remove part of a DNA sequence is to use anexonuclease in combination with DNA amplification to produceunidirectional nested deletions of double stranded DNA clones. Acommercial kit for this purpose is sold under the trade name Exo-Size™(New England Biolabs, Beverly, Mass.). Briefly, this procedure entailsincubating exonuclease III with DNA to progressively remove nucleotidesin the 3′ to 5′ direction at 5′ overhangs, blunt ends or nicks in theDNA template. However, exonuclease III is unable to remove nucleotidesat 3′,4-base overhangs. Timed digests of a clone with this enzymeproduces unidirectional nested deletions.

The entire promoter sequence or portions thereof can be used as a probecapable of specifically hybridizing to corresponding promoter sequences.To achieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length, and most preferably at least about 20nucleotides in length. Such probes can be used to amplify correspondingpromoter sequences from a chosen organism by the well-known process ofpolymerase chain reaction (PCR). This technique can be used to isolateadditional promoter sequences from a desired organism or as a diagnosticassay to determine the presence of the promoter sequence in an organism.Examples include hybridization screening of plated DNA libraries (eitherplaques or colonies; see, e.g., Innis, et al., (1990) PCR Protocols, AGuide to Methods and Applications, eds., Academic Press).

The stress-induced regulatory elements disclosed in the presentinvention, as well as variants and fragments thereof, are useful in thegenetic manipulation of any plant when operably linked with an isolatednucleotide sequence of interest whose expression is to be controlled toachieve a desired phenotypic response.

By “operably linked” is intended a functional linkage between a promoterand a second sequence, wherein the promoter sequence initiates andmediates transcription of the DNA sequence corresponding to the secondsequence. The expression cassette will include a regulatory sequence ofthe invention operably linked to at least one sequence of interest.

In one typical embodiment, in the context of an over expressioncassette, operably linked means that the nucleotide sequences beinglinked are contiguous and, where necessary to join two or more proteincoding regions, contiguous and in the same reading frame. In the casewhere an expression cassette contains two or more protein coding regionsjoined in a contiguous manner in the same reading frame, the encodedpolypeptide is herein defined as a “heterologous polypeptide” or a“chimeric polypeptide” or a “fusion polypeptide”. The cassette mayadditionally contain at least one additional coding sequence to beco-transformed into the organism. Alternatively, the additional codingsequence(s) can be provided on multiple expression cassettes.

The regulatory elements of the invention can be operably linked to theisolated nucleotide sequence of interest in any of several ways known toone of skill in the art. The isolated nucleotide sequence of interestcan be inserted into a site within the genome which is 3′ to thepromoter of the invention using site specific integration as describedin U.S. Pat. No. 6,187,994 herein incorporated in its entirety byreference.

The regulatory elements of the invention can be operably linked inexpression cassettes along with isolated polynucleotide sequences ofinterest for expression in the plant. Such an expression cassette isprovided with a plurality of restriction sites for insertion of thenucleotide sequence of interest under the transcriptional control of theregulatory elements.

The regulatory elements of the invention can be used for directingexpression of a sequence in plant tissues. This can be achieved byincreasing expression of endogenous or exogenous products.Alternatively, the results can be achieved by providing for a reductionof expression of one or more endogenous products, particularly enzymesor cofactors. This down regulation can be achieved through manydifferent approaches known to one skilled in the art, includingantisense, cosuppression, use of hairpin formations, or others, anddiscussed infra. It is recognized that the regulatory elements may beused with their native or other coding sequences to increase or decreaseexpression of an operably linked sequence in the transformed plant orseed.

General categories of genes of interest for the purposes of the presentinvention include for example, those genes involved in information, suchas zinc fingers; those involved in communication, such as kinases; andthose involved in housekeeping, such as heat shock proteins. Morespecific categories of transgenes include genes encoding importanttraits for agronomics, insect resistance, disease resistance, herbicideresistance, and grain characteristics. Still other categories oftransgenes include genes for inducing expression of exogenous productssuch as enzymes, cofactors, and hormones from plants and othereukaryotes as well as prokaryotic organisms.

Modifications that affect grain traits include increasing the content ofoleic acid, or altering levels of saturated and unsaturated fatty acids.Likewise, the level of proteins, particularly modified proteins thatimprove the nutrient value of the plant, can be increased. This isachieved by the expression of such proteins having enhanced amino acidcontent.

Increasing the levels of lysine and sulfur-containing amino acids may bedesired as well as the modification of starch type and content in theseed. Hordothionin protein modifications are described in WO 9416078filed Apr. 10, 1997; WO 9638562 filed Mar. 26, 1997; WO 9638563 filedMar. 26, 1997 and U.S. Pat. No. 5,703,409 issued Dec. 30, 1997. Anotherexample is lysine and/or sulfur-rich root protein encoded by the soybean2S albumin described in WO 9735023 filed Mar. 20, 1996, and thechymotrypsin inhibitor from barley, Williamson, et al., (1987) Eur. J.Biochem. 165:99-106.

Agronomic traits can be improved by altering expression of genes thataffect the response of root, plant or seed growth and development duringenvironmental stress, Cheikh-N, et al., (1994) Plant Physiol.106(1):45-51, and genes controlling carbohydrate metabolism to reducekernel abortion in maize, Zinselmeier, et al., (1995) Plant Physiol.107(2):385-391.

It is recognized that any gene of interest, including the native codingsequence, can be operably linked to the regulatory elements of theinvention and expressed in the plant.

By way of illustration, without intending to be limiting, are examplesof the types of genes which can be used in connection with theregulatory sequences of the invention.

1. Transgenes that Confer Resistance to Insects or Disease and thatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., (1994) Science266:789 (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., (1993) Science 262:1432 (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);Mindrinos, et al., (1994) Cell 78:1089 (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae); McDowell and Woffenden, (2003)Trends Biotechnol. 21(4):178-83 and Toyoda, et al., (2002) TransgenicRes. 11 (6):567-82. A plant resistant to a disease is one that is moreresistant to a pathogen as compared to the wild type plant.

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser, et al.,(1986) Gene 48:109, who disclose the cloning and nucleotide sequence ofa Bt delta-endotoxin gene. Moreover, DNA molecules encodingdelta-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), for example, under ATCC Accession Numbers40098, 67136, 31995 and 31998. Other examples of Bacillus thuringiensistransgenes being genetically engineered are given in the followingpatents and patent applications and hereby are incorporated by referencefor this purpose: U.S. Pat. Nos. 5,188,960; 5,689,052; 5,880,275; PCTApplication Numbers WO 91/14778; WO 99/31248; WO 01/12731; WO 99/24581;WO 97/40162 and U.S. patent application Ser. Nos. 10/032,717; 10/414,637and 10/606,320.

(C) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., (1990) Nature 344:458, of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(D) An insect-specific peptide which, upon expression, disrupts thephysiology of the affected pest. For example, see the disclosures ofRegan, (1994) J. Biol. Chem. 269:9 (expression cloning yields DNA codingfor insect diuretic hormone receptor); Pratt, et al., (1989) Biochem.Biophys. Res. Comm. 163:1243 (an allostatin is identified in Diplopterapuntata); Chattopadhyay, et al., (2004) Critical Reviews in Microbiology30(1):33-54; Zjawiony, (2004) J Nat Prod 67(2):300-310; Carlini andGrossi-de-Sa, (2002) Toxicon 40(11):1515-1539; Ussuf, et al., (2001)Curr Sci. 80(7):847-853; and Vasconcelos and Oliveira (2004) Toxicon44(4):385-403. See also, U.S. Pat. No. 5,266,317 to Tomalski, et al.,who disclose genes encoding insect-specific toxins.

(E) An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxycinnamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

(F) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase or a glucanase, whether natural or synthetic. See, PCTApplication Number WO 93/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC under Accession Numbers 39637 and 67152. See also, Kramer, etal., (1993) Insect Biochem. Molec. Biol. 23:691, who teach thenucleotide sequence of a cDNA encoding tobacco hookworm chitinase; andKawalleck, et al., (1993) Plant Molec. Biol. 21:673, who provide thenucleotide sequence of the parsley ubi4-2 polyubiquitin gene; U.S.application Ser. Nos. 10/389,432, 10/692,367 and U.S. Pat. No.6,563,020.

(G) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., (1994) Plant Molec. Biol. 24:757, ofnucleotide sequences for mung bean calmodulin cDNA clones; and Griess,et al., (1994) Plant Physiol. 104:1467, who provide the nucleotidesequence of a maize calmodulin cDNA clone.

(H) A hydrophobic moment peptide. See, PCT Application Number WO95/16776 and U.S. Pat. No. 5,580,852 (disclosure of peptide derivativesof Tachyplesin which inhibit fungal plant pathogens) and PCT ApplicationNumber WO 95/18855 and U.S. Pat. No. 5,607,914 (teaches syntheticantimicrobial peptides that confer disease resistance).

(I) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes, et al., (1993) Plant Sci. 89:43,of heterologous expression of a cecropin-beta lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(J) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See, Beachy, et al., (1990) Ann. Rev.Phytopathol. 28:451. Coat protein-mediated resistance has been conferredupon transformed plants against alfalfa mosaic virus, cucumber mosaicvirus, tobacco streak virus, potato virus X, potato virus Y, tobaccoetch virus, tobacco rattle virus and tobacco mosaic virus. Id.

(K) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.,Taylor, et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-microbe Interactions (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(L) A virus-specific antibody. See, for example, Tavladoraki, et al.(1993), Nature 366:469, who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(M) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo alpha-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-alpha-1,4-D-galacturonase. See, Lamb,et al., (1992) Bio/Technology 10:1436. The cloning and characterizationof a gene which encodes a bean endopolygalacturonase-inhibiting proteinis described by Toubart, et al., (1992) Plant J. 2:367.

(N) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., (1992) Bio/Technology 10:305, have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, (1995) Current Biology,5(2):128-131; Pieterse and Van Loon (2004) Curr. Opin. Plant Bio.7(4):456-64 and Somssich, (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, (1993) Pl. Physiol.101:709-712; Parijs, et al., (1991) Planta 183:258-264; and Bushnell, etal., (1998) Can. J. of Plant Path. 20(2):137-149. Also see, U.S. patentapplication Ser. No. 09/950,933.

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see, U.S. Pat. No. 5,792,931.

(R) Cystatin and cysteine proteinase inhibitors. See, U.S. patentapplication Ser. No. 10/947,979.

(S) Defensin genes. See, PCT Application Number WO 03/000863 and U.S.patent application Ser. No. 10/178,213.

(T) Genes conferring resistance to nematodes. See, PCT ApplicationNumber WO 03/033651 and Urwin, et al., (1998) Planta 204:472-479;Williamson (1999) Curr Opin Plant Bio. 2(4):327-31.

(U) Genes that confer resistance to Phytophthora Root Rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes.See, for example, Shoemaker, et al., Phytophthora Root Rot ResistanceGene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif.(1995).

(V) Genes that confer resistance to Brown Stem Rot, such as described inU.S. Pat. No. 5,689,035.

2. Transgenes that Confer Resistance to a Herbicide Such as:

(A) An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., (1988) EMBO J. 7:1241; and Miki, et al., (1990) Theor. Appl. Genet.80:449, respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107;5,928,937 and 5,378,824; and PCT Application Number WO 96/33270.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. U.S. Pat. No. 5,627,061 toBarry, et al., also describes genes encoding EPSPS enzymes. See also,U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497; 5,804,425;5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835;5,866,775; 6,225,114; 6,130,366; 5,310,667; 4,535,060; 4,769,061;5,633,448; 5,510,471; Re. 36,449; RE 37,287 E and 5,491,288; andinternational publication numbers EP1173580; WO 01/66704; EP1173581 andEP1173582. Glyphosate resistance is also imparted to plants that expressa gene that encodes a glyphosate oxido-reductase enzyme as describedmore fully in U.S. Pat. Nos. 5,776,760 and 5,463,175. In additionglyphosate resistance can be imparted to plants by the over expressionof genes encoding glyphosate N-acetyltransferase. See, for example, U.S.patent application Ser. No. 10/427,692. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European Patent Application Number 0 333 033 toKumada, et al., and U.S. Pat. No. 4,975,374 to Goodman, et al., disclosenucleotide sequences of glutamine synthetase genes which conferresistance to herbicides such as L-phosphinothricin. The nucleotidesequence of a phosphinothricin-acetyl-transferase gene is provided inEuropean Patent Number 0 242 246 and 0 242 236 to Leemans, et al. DeGreef, et al., (1989) Bio/Technology 7:61, describe the production oftransgenic plants that express chimeric bar genes coding forphosphinothricin acetyl transferase activity. See also, U.S. Pat. Nos.5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236;5,648,477; 5,646,024; 6,177,616 and 5,879,903. Exemplary genesconferring resistance to phenoxy proprionic acids and cycloshexones,such as sethoxydim and haloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3genes described by Marshall, et al., (1992) Theor. Appl. Genet. 83:435.

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla, et al.,(1991) Plant Cell 3:169, describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNumbers 53435, 67441 and 67442. Cloning and expression of DNA coding fora glutathione S-transferase is described by Hayes, et al., (1992)Biochem. J. 285:173.

(D) Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants (see, e.g., Hattori, et al., (1995)Mol Gen Genet. 246:419). Other genes that confer resistance toherbicides include: a gene encoding a chimeric protein of rat cytochromeP4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al.,(1994) Plant Physiol. 106:17), genes for glutathione reductase andsuperoxide dismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687,and genes for various phosphotransferases (Datta, et al., (1992) PlantMol Biol 20:619).

(E) Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837 and5,767,373; and international publication number WO 01/12825.

3. Transgenes That Confer pr Contribute To an Altered GrainCharacteristic, Such As:

(A) Altered fatty acids, for example, by

-   -   (1) Down-regulation of stearoyl-ACP desaturase to increase        stearic acid content of the plant. See, Knultzon, et al., (1992)        Proc. Natl. Acad. Sci. USA 89:2624 and PCT Application Number WO        99/64579 (Genes for Desaturases to Alter Lipid Profiles in        Corn),    -   (2) Elevating oleic acid via FAD-2 gene modification and/or        decreasing linolenic acid via FAD-3 gene modification (see, U.S.        Pat. Nos. 6,063,947; 6,323,392; 6,372,965 and PCT Application        Number WO 93/11245),    -   (3) Altering conjugated linolenic or linoleic acid content, such        as in PCT application Number WO 01/12800,    -   (4) Altering LEC1, AGP, Dek1, Superal1, mi1ps, various Ipa genes        such as Ipa1, Ipa3, hpt or hggt. For example, see, PCT        Application Numbers WO 02/42424, WO 98/22604, WO 03/011015, U.S.        Pat. No. 6,423,886, U.S. Pat. No. 6,197,561, U.S. Pat. No.        6,825,397, US Patent Application Publication Number        2003/0079247, US Patent Application Publication Number        2003/0204870, PCT Application Numbers WO 02/057439, WO 03/011015        and Rivera-Madrid, et. al., (1995) Proc. Natl. Acad. Sci.        92:5620-5624.

(B) Altered phosphorus content, for example, by the

-   -   (1) Introduction of a phytase-encoding gene. This would enhance        breakdown of phytate, adding more free phosphate to the        transformed plant.

For example, see, Van Hartingsveldt, et al., (1993) Gene 127:87, for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

-   -   (2) Up-regulation of a gene that reduces phytate content. In        maize, this, for example, could be accomplished, by cloning and        then re-introducing DNA associated with one or more of the        alleles, such as the LPA alleles, identified in maize mutants        characterized by low levels of phytic acid, such as in Raboy, et        al., (1990) Maydica 35:383 and/or by altering inositol kinase        activity as in PCT Application Number WO 02/059324, US Patent        Application Publication Number 2003/0009011, PCT Application        Number WO 03/027243, US Patent Application Publication Number        2003/0079247, PCT Application Number WO 99/05298, U.S. Pat. No.        6,197,561, U.S. Pat. No. 6,291,224, U.S. Pat. No. 6,391,348, PCT        Application Number WO 2002/059324, US Patent Application        Publication Number 2003/0079247, PCT Application Numbers WO        98/45448, WO 99/55882, WO 01/04147.

(C) Altered carbohydrates effected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or a genealtering thioredoxin. (See, U.S. Pat. No. 6,531,648). See, Shiroza, etal., (1988) J. Bacteriol. 170:810 (nucleotide sequence of Streptococcusmutans fructosyltransferase gene); Steinmetz, et al., (1985) Mol. Gen.Genet. 200:220 (nucleotide sequence of Bacillus subtilis levansucrasegene); Pen, et al., (1992) Bio/Technology 10:292 (production oftransgenic plants that express Bacillus licheniformis alpha-amylase);Elliot, et al., (1993) Plant Molec. Biol. 21 515 (nucleotide sequencesof tomato invertase genes); Søgaard, et al., (1993) J. Biol. Chem.268:22480 (site-directed mutagenesis of barley alpha-amylase gene); andFisher, et al., (1993) Plant Physiol. 102:1045 (maize endosperm starchbranching enzyme II), WO Application Number 99/10498 (improveddigestibility and/or starch extraction through modification ofUDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref1, HCHL, C4H); U.S. Pat.No. 6,232,529 (method of producing high oil seed by modification ofstarch levels (AGP)). The fatty acid modification genes mentioned abovemay also be used to affect starch content and/or composition through theinterrelationship of the starch and oil pathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see, U.S. Pat. No. 6,787,683,US Patent Application Publication Number 2004/0034886 and PCTApplication Number WO 00/68393 involving the manipulation of antioxidantlevels through alteration of a phytl prenyl transferase (ppt), WOApplication Number 03/082899 through alteration of a homogentisategeranyl geranyl transferase (hggt).

(E) Altered essential seed amino acids. For example, see, U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), PCT Application Number WO 99/40209 (alteration of aminoacid compositions in seeds), PCT Application Number WO 99/29882 (methodsfor altering amino acid content of proteins), U.S. Pat. No. 5,850,016(alteration of amino acid compositions in seeds), PCT Application NumberWO 98/20133 (proteins with enhanced levels of essential amino acids),U.S. Pat. No. 5,885,802 (high methionine), U.S. Pat. No. 5,885,801 (highthreonine), U.S. Pat. No. 6,664,445 (plant amino acid biosyntheticenzymes), U.S. Pat. No. 6,459,019 (increased lysine and threonine), U.S.Pat. No. 6,441,274 (plant tryptophan synthase beta subunit), U.S. Pat.No. 6,346,403 (methionine metabolic enzymes), U.S. Pat. No. 5,939,599(high sulfur), U.S. Pat. No. 5,912,414 (increased methionine), PCTApplication Number WO 98/56935 (plant amino acid biosynthetic enzymes),PCT Application Number WO 98/45458 (engineered seed protein havinghigher percentage of essential amino acids), PCT Application Number WO98/42831 (increased lysine), U.S. Pat. No. 5,633,436 (increasing sulfuramino acid content), U.S. Pat. No. 5,559,223 (synthetic storage proteinswith defined structure containing programmable levels of essential aminoacids for improvement of the nutritional value of plants), PCTApplication Number WO 96/01905 (increased threonine), PCT ApplicationNumber WO 95/15392 (increased lysine), US Patent Application PublicationNumber 2003/0163838, US Patent Application Publication Number2003/0150014, US Patent Application Publication Number 2004/0068767,U.S. Pat. No. 6,803,498, PCT Application Number WO 01/79516, and PCTApplication Number WO 00/09706 (Ces A: cellulose synthase), U.S. Pat.No. 6,194,638 (hemicellulose), U.S. Pat. No. 6,399,859 and US PatentApplication Publication Number 2004/0025203 (UDPGdH), U.S. Pat. No.6,194,638 (RGP).

4. Genes that Control Male-Sterility

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar, et al., and chromosomal translocationsas described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen, et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT (PCT Application Number WO 01/29237).

(B) Introduction of various stamen-specific promoters (PCT ApplicationNumbers WO 92/13956, WO 92/13957).

(C) Introduction of the barnase and the barstar gene (Paul, et al.,(1992) Plant Mol. Biol. 19:611-622).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. No. 5,859,341; U.S. Pat. No. 6,297,426; U.S.Pat. No. 5,478,369; U.S. Pat. No. 5,824,524; U.S. Pat. No. 5,850,014;and U.S. Pat. No. 6,265,640.

5. Genes that Create a Site for Site Specific DNA Integration.

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.For example, see, Lyznik, et al., (2003) “Site-Specific Recombinationfor Genetic Engineering in Plants” Plant Cell Rep 21:925-932 and PCTApplication Number WO 99/25821, which are hereby incorporated byreference. Other systems that may be used include the Gin recombinase ofphage Mu (Maeser, et al., (1991) Mol Gen Genet. 230(1-2):170-6); VickiChandler, The Maize Handbook ch. 118 (Springer-Verlag 1994), the Pinrecombinase of E. coli (Enomoto, et al., 1983) and the R/RS system ofthe pSR1 plasmid (Araki, et al., (1992) J Mol. Biol. 225(1):25-37).

6. Genes that Affect Abiotic Stress Resistance

(including but not limited to modulation of flowering, ear and seeddevelopment, enhancement of nitrogen utilization efficiency, alterednitrogen responsiveness, drought resistance or tolerance, coldresistance or tolerance, and salt resistance or tolerance) and increasedyield under stress.

For example, see, PCT Application Number WO 00/73475 where water useefficiency is altered through alteration of malate; U.S. Pat. No.5,892,009, U.S. Pat. No. 5,965,705, U.S. Pat. No. 5,929,305, U.S. Pat.No. 5,891,859, U.S. Pat. No. 6,417,428, U.S. Pat. No. 6,664,446, U.S.Pat. No. 6,706,866, U.S. Pat. No. 6,717,034, U.S. Pat. No. 6,801,104,PCT Application Numbers WO 2000/060089, WO 2001/026459, WO 2001/035725,WO 2001/034726, WO 2001/035727, WO 2001/036444, WO 2001/036597, WO2001/036598, WO 2002/015675, WO 2002/017430, WO 2002/077185, WO2002/079403, WO 2003/013227, WO 2003/013228, WO 2003/014327, WO2004/031349, WO 2004/076638, WO 98/09521 and WO 99/38977 describinggenes, including CBF genes and transcription factors effective inmitigating the negative effects of freezing, high salinity, and droughton plants, as well as conferring other positive effects on plantphenotype; US Patent Application Publication Number 2004/0148654 and PCTApplication Number WO 01/36596 where abscisic acid is altered in plantsresulting in improved plant phenotype such as increased yield and/orincreased tolerance to abiotic stress; PCT Application Numbers WO2000/006341, WO 04/090143, U.S. patent application Ser. Nos. 10/817,483and 09/545,334 where cytokinin expression is modified resulting inplants with increased stress tolerance, such as drought tolerance,and/or increased yield. Also see, PCT Application Numbers WO 02/02776,WO 2003/052063, JP 2002281975, U.S. Pat. No. 6,084,153, PCT ApplicationNumber WO 0164898, U.S. Pat. No. 6,177,275 and U.S. Pat. No. 6,107,547(enhancement of nitrogen utilization and altered nitrogenresponsiveness). For ethylene alteration, see, US Patent ApplicationPublication Number 2004/0128719, US Patent Application PublicationNumber 2003/0166197 and PCT Application Number WO 2000/32761. For planttranscription factors or transcriptional regulators of abiotic stress,see, e.g., US Patent Application Publication Number 2004/0098764 or USPatent Application Publication Number 2004/0078852.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, nutrient uptake, especially nitrogen uptake by plants,nitrogen use efficiency; drought tolerance and water use efficiency;root strength, and root lodging resistance; soil pest management, cornroot worm resistance can be introduced or introgressed into plants, seee.g., PCT Application Numbers WO 97/49811 (LHY), WO 98/56918 (ESD4), WO97/10339 and U.S. Pat. No. 6,573,430 (TFL), U.S. Pat. No. 6,713,663(FT), PCT Application Numbers WO 96/14414 (CON), WO 96/38560, WO01/21822 (VRN1), WO 00/44918 (VRN2), WO 99/49064 (GI), WO 00/46358(FRI), WO 97/29123, U.S. Pat. Nos. 6,794,560, 6,307,126 (GAI), PCTApplication Numbers WO 99/09174 (D8 and Rht), and WO 2004/076638 and WO2004/031349 (transcription factors).

Commercial traits in plants can be created through the expression ofgenes that alter starch or protein for the production of paper,textiles, ethanol, polymers or other materials with industrial uses.

Means of increasing or inhibiting a protein are well known to oneskilled in the art and, by way of example, may include, transgenicexpression, antisense suppression, co-suppression methods including butnot limited to: RNA interference, gene activation or suppression usingtranscription factors and/or repressors, mutagenesis includingtransposon tagging, directed and site-specific mutagenesis, chromosomeengineering (see, Nobrega, et. al., (2004) Nature 431:988-993),homologous recombination, TILLING (Targeting Induced Local Lesions InGenomes; McCallum, et al., (2000) Nature Biotechnol. 18:455-457), andbiosynthetic competition to manipulate the expression of proteins.

Many techniques for gene silencing are well known to one of skill in theart, including but not limited to knock-outs such as by insertion of atransposable element such as Mu, Vicki Chandler, The Maize Handbook ch.118 (Springer-Verlag 1994) or other genetic elements such as a FRT, Loxor other site specific integration site; RNA interference (Napoli, etal., (1990) Plant Cell 2:279-289; U.S. Pat. No. 5,034,323, Sharp (1999)Genes Dev. 13:139-141, Zamore, et al., (2000) Cell 101:25-33; andMontgomery, et al., (1998) PNAS USA 95:15502-15507); virus-induced genesilencing (Burton, et al., (2000) Plant Cell 12:691-705, and Baulcombe(1999) Curr. Op. Plant Bio. 2:109-113); target-RNA-specific ribozymes(Haseloff, et al., (1988) Nature 334:585-591); hairpin structures(Smith, et al., (2000) Nature 407:319-320; PCT Application Numbers WO99/53050; and WO 98/53083); MicroRNA (Aukerman and Sakai (2003) PlantCell 15:2730-2741); ribozymes (Steinecke, et al., (1992) EMBO J.11:1525, and Perriman, et al., (1993) Antisense Res. Dev. 3:253);oligonucleotide mediated targeted modification (e.g., PCT ApplicationNumbers WO 03/076574 and WO 99/25853); zinc-finger targeted molecules(e.g., PCT Application Numbers WO 01/52620; WO 03/048345; and WO00/42219); and other methods or combinations of the above methods knownto those of skill in the art.

Any method of increasing or inhibiting a protein can be used in thepresent invention. Several examples are outlined in more detail belowfor illustrative purposes.

The nucleotide sequence operably linked to the regulatory elementsdisclosed herein can be an antisense sequence for a targeted gene. (See,e.g., Sheehy, et al., (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.5,107,065; 5,453,566 and 5,759,829). By “antisense sequence” is intendeda sequence that is in inverse orientation to the 5′-to-3′ normalorientation of that nucleotide sequence. When delivered into a plantcell, expression of the antisense DNA sequence prevents normalexpression of the DNA nucleotide sequence for the targeted gene. Theantisense nucleotide sequence encodes an RNA transcript that iscomplementary to and capable of hybridizing with the endogenousmessenger RNA (mRNA) produced by transcription of the DNA nucleotidesequence for the targeted gene. In this case, production of the nativeprotein encoded by the targeted gene is inhibited to achieve a desiredphenotypic response. Thus the regulatory sequences disclosed herein canbe operably linked to antisense DNA sequences to reduce or inhibitexpression of a native protein in the plant.

As noted, other potential approaches to impact expression of proteins inthe plant include traditional co-suppression, that is, inhibition ofexpression of an endogenous gene through the expression of an identicalstructural gene or gene fragment introduced through transformation(Goring, et al., (1991) Proc. Natl. Acad. Sci. USA 88:1770-1774co-suppression; Taylor (1997) Plant Cell 9:1245; Jorgensen (1990) TrendsBiotech. 8(12):340-344; Flavell (1994) PNAS USA 91:3490-3496; Finnegan,et al., (1994) Bio/Technology 12:883-888; and Neuhuber, et al., (1994)Mol. Gen. Genet. 244:230-241). In one example, co-suppression can beachieved by linking the promoter to a DNA segment such that transcriptsof the segment are produced in the sense orientation and where thetranscripts have at least 65% sequence identity to transcripts of theendogenous gene of interest, thereby suppressing expression of theendogenous gene in said plant cell. (See, U.S. Pat. No. 5,283,184). Theendogenous gene targeted for co-suppression may be a gene encoding anyprotein that accumulates in the plant species of interest. For example,where the endogenous gene targeted for co-suppression is the 50 kDgamma-zein gene, co-suppression is achieved using an expression cassettecomprising the 50 kD gamma-zein gene sequence, or variant or fragmentthereof.

Additional methods of suppression are known in the art and can besimilarly applied to the instant invention. These methods involve thesilencing of a targeted gene by spliced hairpin RNA's and similarmethods also called RNA interference and promoter silencing (see, Smith,et al., (2000) Nature 407:319-320, Waterhouse and Helliwell (2003) Nat.Rev. Genet. 4:29-38; Waterhouse, et al., (1998) Proc. Natl. Acad. Sci.USA 95:13959-13964; Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci.USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Phystiol.129:1723-1731; and PCT Application Numbers WO 99/53050; WO 99/49029; WO99/61631; WO 00/49035 and U.S. Pat. No. 6,506,559).

For miRNA interference, the expression cassette is designed to expressan RNA molecule that is modeled on an endogenous gene. The miRNAmolecule encodes an RNA that forms a hairpin structure containing a22-nucleotide sequence that is complementary to the endogenous gene(target sequence). miRNA molecules are highly efficient at inhibitingthe expression of endogenous genes, and the RNA interference they induceis inherited by subsequent generations of plants.

In one embodiment, the polynucleotide to be introduced into the plantcomprises an inhibitory sequence that encodes a zinc finger protein thatbinds to a gene resulting in reduced expression of the gene. Inparticular embodiments, the zinc finger protein binds to a regulatoryregion of the invention. In other embodiments, the zinc finger proteinbinds to a messenger RNA encoding a protein and prevents itstranslation. Methods of selecting sites for targeting by zinc fingerproteins have been described, for example, in U.S. Pat. No. 6,453,242,and methods for using zinc finger proteins to inhibit the expression ofgenes in plants are described, for example, in US Patent ApplicationPublication Number 2003/0037355.

The expression cassette may also include, at the 3′ terminus of theisolated nucleotide sequence of interest, a transcriptional andtranslational termination region functional in plants. The terminationregion can be native with the promoter nucleotide sequence of thepresent invention, can be native with the DNA sequence of interest, orcan be derived from another source.

Any convenient termination regions can be used in conjunction with thepromoter of the invention, and are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau, et al., (1991) Mol. Gen.Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon, et al.,(1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al.,(1989) Nucleic Acids Res. 17:7891-7903; Joshi, et al., (1987) NucleicAcid Res. 15:9627-9639.

The expression cassettes can additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region),Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130;potyvirus leaders, for example, TEV leader (Tobacco Etch Virus),Allison, et al., (1986) “The Nucleotide Sequence of the Coding Region ofTobacco Etch Virus Genomic RNA: Evidence for the Synthesis of a SinglePolyprotein”, Virology 154:9-20; MDMV leader (Maize Dwarf Mosaic Virus);human immunoglobulin heavy-chain binding protein (BiP), Macejak, et al.,(1991) Nature 353:90-94; untranslated leader from the coat protein mRNAof alfalfa mosaic virus (AMV RNA 4), Jobling, et al., (1987) Nature325:622-625); tobacco mosaic virus leader (TMV), Gallie, et al., (1989)Molecular Biology of RNA, pages 237-256; and maize chlorotic mottlevirus leader (MCMV), Lommel, et al., (1991) Virology 81:382-385. Seealso, Della-Cioppa, et al., (1987) Plant Physiology 84:965-968. Thecassette can also contain sequences that enhance translation and/or mRNAstability such as introns.

In those instances where it is desirable to have an expressed product ofan isolated nucleotide sequence directed to a particular organelle,particularly the plastid, amyloplast, or to the endoplasmic reticulum,or secreted at the cell's surface or extracellularly, the expressioncassette can further comprise a coding sequence for a transit peptide.Such transit peptides are well known in the art and include, but are notlimited to: the transit peptide for the acyl carrier protein, the smallsubunit of RUBISCO, plant EPSP synthase, and the like.

In preparing the expression cassette, the various DNA fragments can bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers can be employed to join the DNA fragments,or other manipulations can be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction digests, annealing, and resubstitutions such astransitions and transversions, can be involved.

As noted herein, the present invention provides vectors capable ofexpressing genes of interest under the control of the regulatoryelements of the invention. In general, the vectors should be functionalin plant cells. At times, it may be preferable to have vectors that arefunctional in E. coli (e.g., production of protein for raisingantibodies, DNA sequence analysis, construction of inserts, obtainingquantities of nucleic acids). Vectors and procedures for cloning andexpression in E. coli are discussed in Sambrook, et al. (supra).

The transformation vector, comprising the regulatory sequences of thepresent invention operably linked to an isolated polynucleotide sequencein an expression cassette, can also contain at least one additionalnucleotide sequence for a gene to be cotransformed into the organism.Alternatively, the additional sequence(s) can be provided on anothertransformation vector.

Vectors that are functional in plants can be binary plasmids derivedfrom Agrobacterium. Such vectors are capable of transforming plantcells. These vectors contain left and right border sequences that arerequired for integration into the host (plant) chromosome. At a minimum,between these border sequences is the gene to be expressed under controlof the regulatory elements of the present invention. In one embodiment,a selectable marker and a reporter gene are also included. For ease ofobtaining sufficient quantities of vector, a bacterial origin thatallows replication in E. coli can be used.

Reporter genes can be included in the transformation vectors. Examplesof suitable reporter genes known in the art can be found in, forexample, Jefferson, et al., (1991) in Plant Molecular Biology Manual,ed. Gelvin, et al., (Kluwer Academic Publishers), pp. 1-33; DeWet, etal., (1987) Mol. Cell. Biol. 7:725-737; Goff, et al., (1990) EMBO J.9:2517-2522; Kain, et al., (1995) BioTechniques 19:650-655; and Chiu, etal., (1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissuescan be included in the transformation vectors. These can include genesthat confer antibiotic resistance or resistance to herbicides. Examplesof suitable selectable marker genes include, but are not limited to:genes encoding resistance to chloramphenicol, Herrera Estrella, et al.,(1983) EMBO J. 2:987-992; methotrexate, Herrera Estrella, et al., (1983)Nature 303:209-213; Meijer, et al., (1991) Plant Mol. Biol. 16:807-820;hygromycin, Waldron, et al., (1985) Plant Mol. Biol. 5:103-108; Zhijian,et al., (1995) Plant Science 108:219-227; streptomycin, Jones, et al.,(1987) Mol. Gen. Genet. 210:86-91; spectinomycin, Bretagne-Sagnard, etal., (1996) Transgenic Res. 5:131-137; bleomycin, Hille, et al., (1990)Plant Mol. Biol. 7:171-176; sulfonamide, Guerineau, et al., (1990) PlantMol. Biol. 15:127-136; bromoxynil, Stalker, et al., (1988) Science242:419-423; glyphosate, Shaw, et al., (1986) Science 233:478-481;phosphinothricin, DeBlock, et al., (1987) EMBO J. 6:2513-2518.

Further, when linking a promoter of the invention with a nucleotidesequence encoding a detectable protein, stress-induced expression of alinked sequence can be tracked, thereby providing a useful so-calledscreenable or scorable markers. The expression of the linked protein canbe detected without destroying tissue. More recently, interest hasincreased in utilization of screenable or scorable markers. By way ofexample without limitation, the promoter can be linked with detectablemarkers including a β-glucuronidase, or uidA gene (GUS), which encodesan enzyme for which various chromogenic substrates are known (Jefferson,et al., (1986) Proc. Natl. Acad. Sci. USA 83:8447-8451); chloramphenicolacetyl transferase; alkaline phosphatase; a R-locus gene, which encodesa product that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta, et al., (1988) in ChromosomeStructure and Function, Kluwer Academic Publishers, Appels and Gustafsoneds., pp. 263-282; Ludwig, et al., (1990) Science 247:449); ap-lactamase gene (Sutcliffe, (1978) Proc. Nat'l. Acad. Sci. U.S.A.75:3737), which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylEgene (Zukowsky, et al., (1983) Proc. Nat'l. Acad. Sci. U.S.A. 80:1101),which encodes a catechol dioxygenase that can convert chromogeniccatechols; an α-amylase gene (Ikuta, et al., (1990) Biotech. 8:241); atyrosinase gene (Katz, et al., (1983) J. Gen. Microbiol. 129:2703),which encodes an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone, which in turn condenses to form the easily detectablecompound melanin a green fluorescent protein (GFP) gene (Sheen, et al.,(1995) Plant J. 8(5):777-84); a lux gene, which encodes a luciferase,the presence of which may be detected using, for example, X-ray film,scintillation counting, fluorescent spectrophotometry, low-light videocameras, photon counting cameras or multiwell luminometry (Teeri, etal., (1989) EMBO J. 8:343); DS-RED EXPRESS (Matz, et al., (1999) NatureBiotech. 17:969-973, Bevis, et al., (2002) Nature Biotech 20:83-87,Haas, et al., (1996) Curr. Biol. 6:315-324); Zoanthus sp. yellowfluorescent protein (ZsYellow) that has been engineered for brighterfluorescence (Matz, et al., (1999) Nature Biotech. 17:969-973, availablefrom BD Biosciences Clontech, Palo Alto, Calif., USA, catalog no.K6100-1); and cyan florescent protein (CYP) (Bolte, et al., (2004) J.Cell Science 117:943-54 and Kato, et al., (2002) Plant Physiol129:913-42).

A transformation vector comprising the particular regulatory sequencesof the present invention, operably linked to an isolated polynucleotidesequence of interest in an expression cassette, can be used to transformany plant. In this manner, genetically modified plants, plant cells,plant tissue, and the like can be obtained. Transformation protocols canvary depending on the type of plant or plant cell, i.e., monocot ordicot, targeted for transformation. Suitable methods of transformingplant cells include microinjection, Crossway, et al., (1986)Biotechniques 4:320-334; electroporation, Riggs, et al., (1986) Proc.Natl. Acad. Sci. USA 83:5602-5606; Agrobacterium-mediatedtransformation, see, for example, Townsend, et al., U.S. Pat. No.5,563,055; direct gene transfer, Paszkowski, et al., (1984) EMBO J.3:2717-2722; and ballistic particle acceleration, see, for example,Sanford, et al., U.S. Pat. No. 4,945,050, Tomes, et al., (1995) in PlantCell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg andPhillips (Springer-Verlag, Berlin); and McCabe, et al., (1988)Biotechnology 6:923-926. Also see, Weissinger, et al., (1988) AnnualRev. Genet. 22:421-477; Sanford, et al., (1987) Particulate Science andTechnology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol.87:671-674 (soybean); McCabe, et al., (1988) Bio/Technology 6:923-926(soybean); Datta, et al., (1990) Bio/Technology 8:736-740 (rice); Klein,et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein,et al., (1988) Biotechnology 6:559-563 (maize); Klein, et al., (1988)Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990) Biotechnology8:833-839; Hooydaas-Van Slogteren, et al., (1984) Nature (London)311:763-764; Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The ExperimentalManipulation of Ovule Tissues, ed. G. P. Chapman, et al., (Longman, NewYork), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports9:415-418; and Kaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D. Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou, et al., (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens).

The cells that have been transformed can be grown into plants inaccordance with conventional methods. See, for example, McCormick, etal., (1986) Plant Cell Reports 5:81-84. These plants can then be grownand pollinated with the same transformed strain or different strains.The resulting plant having stress-induced expression of the desiredphenotypic characteristic can then be identified. Two or moregenerations can be grown to ensure that stress-induced expression of thedesired phenotypic characteristic is stably maintained and inherited.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

As the coding regions for ZmCBF1 and ZmCBF2 had previously beenisolated, 5′ regulatory regions were isolated from maize plants viaGenomeWalker™ (Clontech) and cloned. Isolated promoter regions wereanalyzed for cis elements and compared to Arabidopsis and rice CBFpromoters.

Transformation of Maize by Particle Bombardment

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing an expression cassette comprising the ZmCBF1 orZmCBF2 promoter operably linked to a gene of interest. The plasmid alsocomprises a selectable marker gene, for example PAT (Wohlleben, et al.,(1988) Gene 70:25-37), which confers resistance to the herbicideBialaphos. Alternatively, the selectable marker gene is provided on aseparate plasmid. Transformation is performed as follows. Media recipesfollow below.

The ears are husked and surface sterilized in 30% Clorox® bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 5 hoursand then aligned within the 2.5 cm target zone in preparation forbombardment.

A plasmid vector is made which comprises the ZmCBF1 or ZmCBF2 promotersequence operably linked to a gene of interest. This plasmid DNA plusplasmid DNA containing a PAT selectable marker is precipitated onto 1.1μm (average diameter) tungsten pellets using a CaCl₂ precipitationprocedure as follows: 100 μl prepared tungsten particles in water; 10 μl(1 μg) DNA in Tris EDTA buffer (1 μg total DNA); 100 μl 2.5 M CaCl₂;and, 10 μl 0.1 M spermidine.

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

The sample plates are bombarded at level #5 in particle gun #HE35-1 or#HE35-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-5 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored under various conditions and compared tocontrol plants. Marker gene expression is observed to confirmtransformation. Alterations in phenotype, reflecting expression of thegene of interest, are monitored.

Bombardment medium (560Y) comprises 5.0 g/l N6 basal salts (SIGMAC-1516), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,5-D, and 2.88 g/l L-proline(brought to volume with D-1 H₂O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite® gelling agent (added after bringing to volumewith D-I H₂O); and 8.5 mg/l silver nitrate (added after sterilizing themedium and cooling to room temperature). Selection medium (560R)comprises 5.0 g/l N6 basal salts (SIGMA C-1516), 1.0 ml/l Eriksson'sVitamin Mix (1000× SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose,and 2.0 mg/l 2,5-D (brought to volume with D-I H₂O following adjustmentto pH 5.8 with KOH); 3.0 g/l Gelrite® gelling agent (added afterbringing to volume with D-I H₂O); and 0.85 mg/l silver nitrate and 3.0mg/l bialaphos (both added after sterilizing the medium and cooling toroom temperature).

Plant regeneration medium (288J) comprises 5.3 g/l MS salts (GIBCO11117-075), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.50 g/l glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog (1962)Physiol. Plant. 15:573), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite® gellingagent (added after bringing to volume with D-I H₂O); and 1.0 mg/lindoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing themedium and cooling to 60° C.). Hormone-free medium (272V) comprises 5.3g/l MS salts (GIBCO 11117-075), 5.0 ml/l MS vitamins stock solution(0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxineHCL, and 0.50 g/l glycine brought to volume with polished D-I H₂O), 0.1g/1 myo-inositol, and 50.0 g/l sucrose (brought to volume with polishedD-I H₂O after adjusting pH to 5.6); and 6 g/l Bacto™ Agar solidifyingagent (added after bringing to volume with polished D-I H₂O), sterilizedand cooled to 60° C.

Example 2 Transformation and Regeneration of Maize Callus viaAgrobacterium

For Agrobacterium-mediated transformation of maize with the ZmCBF1 orZmCBF2 promoter sequence (SEQ ID NO: 1 or 2) operably linked to a geneof interest, the method of Zhao is employed (U.S. Pat. No. 5,981,840,and PCT Patent Publication WO98/32326, the contents of which are herebyincorporated by reference). The immature embryos are cultured on solidmedium with a selective agent resulting in the selective growth oftransformed cells. The callus is then regenerated into plants, and calligrown on selective medium are cultured on solid medium to regenerate theplants.

The plants are monitored for a modulation in phenotype when compared toan appropriate control plant.

Details of Agrobacterium transformation may be as set out below or asknown to one of skill in the art.

Preparation of Agrobacterium Suspension

Agrobacterium is streaked out from a −80° C. frozen aliquot onto a platecontaining PHI-L medium and cultured at 28° C. in the dark for 3 days.PHI-L media comprises 25 ml/l Stock Solution A, 25 ml/l Stock SolutionB, 450.9 ml/l Stock Solution C and spectinomycin (Sigma Chemicals) addedto a concentration of 50 mg/l in sterile ddH₂O (stock solution A: K₂HPO₄60.0 g/l, NaH₂PO₄ 20.0 g/l, adjust pH to 7.0 w/KOH and autoclaved; stocksolution B: NH₄Cl 20.0 g/l, MgSO₄.7H₂O 6.0 g/l, KCl 3.0 g/l, CaCl₂ 0.20g/l, FeSO₄.7H₂O 50.0 mg/l, autoclaved; stock solution C: glucose 5.56g/l, agar 16.67 g/l (#A-7049, Sigma Chemicals, St. Louis, Mo.) andautoclaved).

The plate can be stored at 4° C. and used usually for about 1 month. Asingle colony is picked from the master plate and streaked onto a platecontaining PHI-M medium [yeast extract (Difco) 5.0 g/l; peptone(Difco)10.0 g/l; NaCl 5.0 g/l; agar (Difco) 15.0 g/l; pH 6.8, containing50 mg/L spectinomycin] and incubated at 28° C. in the dark for 2 days.

Five ml of either PHI-A, [CHU(N6) basal salts (Sigma C-1416) 4.0 g/l,Eriksson's vitamin mix (1000×, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5mg/l (Sigma); 2,4-dichlorophenoxyacetic acid (2,4-D, Sigma) 1.5 mg/l;L-proline (Sigma) 0.69 g/l; sucrose (Mallinckrodt) 68.5 g/l; glucose(Mallinckrodt) 36.0 g/l; pH 5.2] for the PHI basic medium system, orPHI-I [MS salts (GIBCO BRL) 4.3 g/l; nicotinic acid (Sigma) 0.5 mg/l;pyridoxine.HCl (Sigma) 0.5 mg/l; thiamine.HCl 1.0 mg/l; myo-inositol(Sigma) 0.10 g/l; vitamin assay casamino acids (Difco Lab) 1 g/l; 2,4-D1.5 mg/l; sucrose 68.50 g/l; glucose 36.0 g/l; adjust pH to 5.2 w/KOHand filter-sterilize] for the PHI combined medium system and 5 ml of 100mM (3′-5′-Dimethoxy-4′-hydroxyacetophenone, Aldrich chemicals) are addedto a 14 ml Falcon tube in a hood. About 3 full loops (5 mm loop size)Agrobacterium are collected from the plate and suspended in the tube,then the tube is vortexed to make an even suspension. One ml of thesuspension is transferred to a spectrophotometer tube and the OD of thesuspension is adjusted to 0.72 at 550 nm by adding either moreAgrobacterium or more of the same suspension medium, for anAgrobacterium concentration of approximately 0.5×109 cfu/ml to 1×109cfu/ml. The final Agrobacterium suspension is aliquoted into 2 mlmicrocentrifuge tubes, each containing 1 ml of the suspension. Thesuspensions are then used as soon as possible.

Embryo Isolation, Infection and Co-Cultivation

About 2 ml of the same medium (here PHI-A or PHI-I) which is used forthe Agrobacterium suspension is added into a 2 ml microcentrifuge tube.Immature embryos are isolated from a sterilized ear with a sterilespatula (Baxter Scientific Products S1565) and dropped directly into themedium in the tube. A total of about 100 embryos are placed in the tube.The optimal size of the embryos is about 1.0-1.2 mm. The cap is thenclosed on the tube and the tube is vortexed with a Vortex Mixer (BaxterScientific Products S8223-1) for 5 sec. at maximum speed. The medium isremoved and 2 ml of fresh medium are added and the vortexing repeated.All of the medium is drawn off and 1 ml of Agrobacterium suspension isadded to the embryos and the tube is vortexed for 30 sec. The tube isallowed to stand for 5 min. in the hood. The suspension of Agrobacteriumand embryos is poured into a Petri plate containing either PHI-B medium[CHU(N6) basal salts (Sigma C-1416) 4.0 g/l; Eriksson's vitamin mix(1000×, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D1.5 mg/l;L-proline 0.69 g/l; silver nitrate 0.85 mg/l; Gelrite® gelling agent(Sigma) 3.0 g/l; sucrose 30.0 g/l; acetosyringone 100 mM; pH 5.8], forthe PHI basic medium system, or PHI-J medium [MS Salts 4.3 g/l;nicotinic acid 0.50 mg/l; pyridoxine HCl 0.50 mg/l; thiamine.HCl 1.0mg/l; myo-inositol 100.0 mg/l; 2,4-D1.5 mg/l; sucrose 20.0 g/l; glucose10.0 g/l; L-proline 0.70 g/l; MES (Sigma) 0.50 g/l; 8.0 g/l agar (SigmaA-7049, purified) and 100 mM acetosyringone with a final pH of 5.8 forthe PHI combined medium system. Any embryos left in the tube aretransferred to the plate using a sterile spatula. The Agrobacteriumsuspension is drawn off and the embryos placed axis side down on themedia. The plate is sealed with Parafilm® tape or Pylori VegetativeCombine Tape (product named “E.G.CUT” and available in 18 mm×50 msections; Kyowa Ltd., Japan) and is incubated in the dark at 23-25° C.for about 3 days of co-cultivation.

Resting, Selection and Regeneration Steps

For the resting step, all of the embryos are transferred to a new platecontaining PHI-C medium [CHU(N6) basal salts (Sigma C-1416) 4.0 g/l;Eriksson's vitamin mix (1000× Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5mg/l; 2.4-D 1.5 mg/l; L-proline 0.69 g/l; sucrose 30.0 g/l; MES buffer(Sigma) 0.5 g/l; agar (Sigma A-7049, purified) 8.0 g/l; silver nitrate0.85 mg/l; carbenicillin 100 mg/l; pH 5.8]. The plate is sealed withParafilm® or Pylon tape and incubated in the dark at 28° C. for 3-5days.

Longer co-cultivation periods may compensate for the absence of aresting step since the resting step, like the co-cultivation step,provides a period of time for the embryo to be cultured in the absenceof a selective agent. Those of ordinary skill in the art can readilytest combinations of co-cultivation and resting times to optimize orimprove the transformation

For selection, all of the embryos are then transferred from the PHI-Cmedium to new plates containing PHI-D medium, as a selection medium,[CHU(N6) basal salts (SIGMA C-1416) 4.0 g/l; Eriksson's vitamin mix(1000×, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l;L-proline 0.69 g/l; sucrose 30.0 g/l; MES buffer 0.5 g/l; agar (SigmaA-7049, purified) 8.0 g/l; silver nitrate 0.85 mg/l; carbenicillin (ICN,Costa Mesa, Calif.) 100 mg/l; bialaphos (Meiji Seika K. K., Tokyo,Japan) 1.5 mg/l for the first two weeks followed by 3 mg/l for theremainder of the time.; pH 5.8] putting about 20 embryos onto eachplate.

The plates are sealed as described above and incubated in the dark at28° C. for the first two weeks of selection. The embryos are transferredto fresh selection medium at two-week intervals. The tissue issubcultured by transferring to fresh selection medium for a total ofabout 2 months. The herbicide-resistant calli are then “bulked up” bygrowing on the same medium for another two weeks until the diameter ofthe calli is about 1.5-2 cm.

For regeneration, the calli are then cultured on PHI-E medium [MS salts4.3 g/l; myo-inositol 0.1 g/l; nicotinic acid 0.5 mg/l, thiamine.HCl 0.1mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l, Zeatin 0.5 mg/l,sucrose 60.0 g/l, Agar (Sigma, A-7049) 8.0 g/l, Indoleacetic acid (IAA,Sigma) 1.0 mg/l, Abscisic acid (ABA, Sigma) 0.1 mM, Bialaphos 3 mg/l,carbenicillin 100 mg/l adjusted to pH 5.6] in the dark at 28° C. for 1-3weeks to allow somatic embryos to mature. The calli are then cultured onPHI-F medium (MS salts 4.3 g/l; myo-inositol 0.1 g/l; Thiamine.HCl 0.1mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l, nicotinic acid 0.5mg/l; sucrose 40.0 g/l; Gelrite® gelling agent 1.5 g/l; pH 5.6] at 25°C. under a daylight schedule of 16 hrs. light (270 uE m-2sec-1) and 8hrs. dark until shoots and roots are developed. Each small plantlet isthen transferred to a 25×150 mm tube containing PHI-F medium and isgrown under the same conditions for approximately another week. Theplants are transplanted to pots with soil mixture in a greenhouse.Transformation events are determined at the callus stage or regeneratedplant stage.

Ability of the ZmCBF1 or ZmCBF2 promoter to drive expression in maize isconfirmed by marker gene detection in plant tissue.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. All referencescited are incorporated herein by reference.

1. An isolated nucleic acid molecule comprising a polynucleotide whichhas promoter activity and is a sequence selected from the groupconsisting of: a) SEQ ID NO: 2 or SEQ ID NO: 1; b) at least 500contiguous nucleotides of SEQ ID NO: 2 or SEQ ID NO: 1; c) variantsequences at least 95% identical and retaining all regulatory elementsidentified in SEQ ID NO: 2 or SEQ ID NO: 1; and d) the complement of SEQID NO: 2 or SEQ ID NO:
 1. 2. An expression cassette comprising thepolynucleotide of claim 1 operably linked to a polynucleotide ofinterest.
 3. A vector comprising the expression cassette of claim
 2. 4.A plant cell having stably incorporated into its genome the expressioncassette of claim
 2. 5. The plant cell of claim 4, wherein said plantcell is from a monocot.
 6. The plant cell of claim 5, wherein saidmonocot is maize, barley, wheat, oat, rye, sorghum or rice.
 7. A planthaving stably incorporated into its genome the expression cassette ofclaim
 2. 8. The plant of claim 7, wherein said plant is a monocot. 9.The plant of claim 8, wherein said monocot is maize, barley, wheat, oat,rye, sorghum or rice.
 10. A transgenic seed of the plant of claim
 7. 11.The plant of claim 7, wherein the polynucleotide of interest encodes agene product that confers drought tolerance or cold tolerance or salttolerance.
 12. The plant of claim 7, wherein the polynucleotide ofinterest encodes a polypeptide involved in nutrient uptake, nitrogen useefficiency, root strength, root lodging resistance, soil pestmanagement, corn rootworm resistance, carbohydrate metabolism, proteinmetabolism, fatty acid metabolism or phytohormone biosynthesis.
 13. Amethod for expressing a first polynucleotide in a plant, said methodpromoter and a first polynucleotide operably linked thereto, whereinsaid promoter comprises a second polynucleotide which has promoteractivity and is a sequence selected from the group consisting of: a) SEQID NO: 2 or SEQ ID NO: 1; b) at least 500 contiguous nucleotides of SEQID NO:1 or SEQ ID NO: 2; c) variant sequences at least 95% identical andretaining all regulatory elements identified in SEQ ID NO: 2 or SEQ IDNO: 1; and d) the complement of SEQ ID NO: 2 or SEQ ID NO:
 1. 14. Themethod of claim 13, wherein said plant is a monocot.
 15. The method ofclaim 14, wherein said monocot is maize, barley, wheat, oat, rye,sorghum or rice.
 16. The method of claim 13, wherein said firstpolynucleotide encodes a gene product that confers drought tolerance orcold tolerance or salt tolerance.
 17. The method of claim 13, whereinsaid first polynucleotide encodes a polypeptide involved in nutrientuptake, nitrogen use efficiency, root strength, root lodging resistance,soil pest management, corn rootworm resistance, carbohydrate metabolism,protein metabolism, fatty acid metabolism or phytohormone biosynthesis.18. A method for expressing a first polynucleotide in a plant cell, saidmethod comprising introducing into a plant cell an expression cassettecomprising a promoter and a first polynucleotide operably linkedthereto, wherein said promoter comprises a second polynucleotide whichhas promoter activity and is a sequence selected from the groupconsisting of: a) SEQ ID NO: 2 or SEQ ID NO: 1 b) at least 500contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2; c) a sequence atleast 95% identical to, and retaining all regulatory elements identifiedin SEQ ID NO: 2 or SEQ ID NO: 1; and d) the complement of SEQ ID NO: 2or SEQ ID NO:
 1. 19. The method of claim 18, wherein said firstpolynucleotide encodes a gene product that confers drought tolerance orcold tolerance or salt tolerance.
 20. The method of claim 18, whereinsaid first polynucleotide encodes a polypeptide involved in nutrientuptake, nitrogen use efficiency, root strength, root lodging resistance,soil pest management, corn root worm resistance, carbohydratemetabolism, protein metabolism, fatty acid metabolism, or phytohormonebiosynthesis.